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Vol. 17, Issue 8, 3378-3385, August 2006

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The Detection of Micromolar Pericellular ATP Pool on Lymphocyte Surface by Using Lymphoid Ecto-Adenylate Kinase as Intrinsic ATP Sensor

Gennady G. Yegutkin^*, Andrey Mikhailov, Sergei S. Samburski^*, and Sirpa Jalkanen^*

*MediCity Laboratory and Department of Medical Microbiology, Turku University and National Public Health Institute, FIN-20520 Turku, Finland; and Turku Centre for Biotechnology, University of Turku/Åbo Akademi University, FIN-20521 Turku, Finland

Submitted October 28, 2005; Revised April 17, 2006; Accepted May 10, 2006
Monitoring Editor: Guido Guidotti

Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [³H]AMP or [-³²P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous -phosphate–donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y₂-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Gennady G. Yegutkin ( gennady.yegutkin{at}utu.fi)

Abbreviations used: AK, adenylate kinase; APCP, ,-methylene-ADP; Ap₅A, diadenosine pentaphosphate; CTB, cholera toxin subunit B; NTPDase, nucleoside triphosphate diphosphohydrolase; PBL, peripheral blood lymphocytes; TLC, thin-layer chromatography

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